Endophytes such as the fungus Clonostachys rosea formerly called Gliocladium roseum, have been most commonly described in the literature as a “site occupier” i.e. endophytes “or fungi or bacteria that form symptom less infections, for part or all of their life cycle within healthy leaves and stems of plants” (Definition by Hawksworth et al, 1995).
Sample data on endophytes such as the fungus Clonostachys rosea or Gliocladium roseum (as it was formerly known), indicate that the fungus occurs worldwide in a variety of soils e.g. Europe, North America, Latin America, etc. However not all strains of Clonostachys rosea are the same in morphology, reproductively or have the same genetic material. Strain 88-710 described in this invention is unique reproductively to the fungus species Clonostachyus. 
The background for the classification of Clonostachys rosea is described in the reference i.e. (Mycologia: Vol. 91, No. 2, pp. 365-385.1999. Hans-Josef Schroers, Gary J. Samuels, Keith A. Seifert, and Walter Gams.
Because the common soil fungus and mycoparasite Gliocladium roseum differs from the type species of Gliocladium, G. penicillioides, in morphology, ecology, teleomorph, and DNA sequence data, it is classified in a separate genus, Clonostachys. Penicillium roseum is the oldest available name for G. roseum and is recombined as C. rosea. Penicillium roseum, described from potato in Germany, is neotypified by a conidial isolate originating from a fungal substratum in European soil. By choosing this strain as neotype for P. roseum the epithet is formally linked to the common soil fungus used in the biocontrol of fungal plant pathogens. The anamorph of Bionectria ochroleuca (Hypocreales) is morphologically indistinguishable from C. rosea; both morphs are redescribed. Bionectria is generically distinct from Nectria s. s. and is the appropriate genus for species of the Nectria ochroleuca group. The anamorph genus Gliocladium s. s. is associated with teleomorphs in Sphaerostilbella and Hypocrea series Pallidae. With the separation of Clonostachys from Gliocladium and Bionectria from Nectria the generic classification reflects natural relationships. A generic circumscription is proposed for Clonostachys and compared with Gliocladium. Nectriopsis sporangiicola and Roumegueriella rufula are related to Bionectria but have distinct Gliocladium-like anamorphs. Based on morphological features, Rhopalocladium myxophilum gen. et sp. nov. is proposed for the anamorph of N. sporangiicola. The anamorph of Roumegueriella rufula is generally found in association with the teleomorph and is referred to as Gliocladium-like.
Not all endophytes respond to plants in the same way. Endophytes are known to infect healthy plants e.g. via wounds e.g. mechanical damage caused by insects or diseases and in some instances research has documented certain endophytes to provide some measure of plant disease or control of insect pests.
However, little or no documentation has been provided that show endophytes to act as inoculants to promote plant health, growth or vigor and capable of providing “natural inoculant induced plant resistance (IIR)” to environmental stresses. Moreover, little or no information has been provided to show that specific endophytes such as the strain 88-710 of Clonostachys rosea when acting as an inoculant can also co-exist with other beneficial fungi, bacteria or viruses to provide additive or stimulated growth benefits.
For example, there is no known data or information that demonstrates that a fungus esp. an endophyte e.g. Clonostachys rosea strain 88-710 can act as an inoculant to stimulate the production of nitrogen fixing nodules on legumes e.g. soybeans. Until this invention, it was thought that only naturally occurring bacteria e.g. Rhizobium were capable of producing and stimulating the production of nitrogen fixing nodules on legume plants and reducing dependency on chemical fertilizers. This invention teaches that Clonostachyus rosea strain 88-710 can act as an inoculant to stimulate and be additive (beneficially interactive with nitrogen fixing bacteria) in the production of nitrogen fixing nodules on legumes e.g. soybeans
This invention describes new examples/traits about endophytes such as the strain 88-710 of Clonostachys rosea that are novel, inventive, useful that teaches something new. The fungus called Clonostachys rosea (C. rosea) although it occurs in virtually all parts of the world, in various strains/forms and in various climate zones, it has most often been characterized as a closely (morphological) counterpart of Penicillium with slimy/sticky conidia. The role and function of Penicillium spp. as inoculants, is well documented but the inoculant function and mode of action of its closely related relative i.e. the endophyte C. rosea has not been documented, quantified or demonstrated until this invention.
Until recently, the role or mode of action of endophytes such as C. rosea was not fully understood and it was thought that all strains of C. rosea exhibited only biocontrol modes of action; most such studies were conducted in the presence of plant diseases and not in the absence or near absence of plant diseases. The data appeared to only measure or indicate that C. rosea or Glioclaidium roseum (as it was previously known) may control various fungal infections of plants as a biocontrol agent e.g. mycoparasite or hyperparasite by exuding antibiotics, metabolites or mycoparasitic enzymes.
However, as this invention shows, endophytes such as C. rosea, strain 88-710 do not directly control fungal or bacterial diseases nor do they have a direct mitigating role of controlling plant pests i.e. diseases or insects, mites, etc. by exuding antibiotics, toxic metabolites or enzymes. Rather as the novelty of this invention will demonstrate, is that C. rosea acts as a true inoculant on plant tissues to promote plant health, size, root growth or increases yield and also can aid in reducing or eliminating the use of chemical fungicides while as it imparts natural induced plant resistance with suitable formulations to environmental stresses such as plant diseases, moisture reduction, insects and mites. It does this mainly by having the ability to rapidly colonise plant tissue (living or senescing tissue), imparting resistance to the plant while occupying the tissue thus denying such tissue to infection by disease organisms, and other stresses while assisting the plant to uptake nutrients.
The current invention also describes C. rosea strain 88-710 as providing plant health benefits to plants irrespective of whether a pathogen is present, hence true non-biocontrol features.
The mode of action of C. rosea strain 88-710 thus appears instead to be one of it ability to provide a rapid first presence as an “inoculant site occupier” to leaves, stems, and roots thereby promoting plant vigor, plant health, growth and stress reduction such as the prevention of root biomass loss during stem trimming. This ability to help plants retain good root mass/growth helps plants ward off or re-cover from the shock of trimming (bonsai effect) resulting in faster re-generation of stem growth and better utilization of nutrients. It also acts in the same way to overcome other stresses to plants whether those stresses are environmental or cultural.
Moreover, endophytes such as C. rosea, strain 88-710 are thought to work by enhancing the solubility/availability of plant nutrients e.g. phosphorus (Reference: Tilak, K. V. B. R et al “Diversity of Plant Growth and Soil Health Supporting Bacteria”, July 2005 Current Science Vol 89 N01). This novel invention will show that the strain 88-710 of Clonostachyus rosea acts to enhance nitrogen, phosphorus, and potassium uptake as well micronutrients in plants and can act complementary/additive to nitrogen fixing Rhizobium bacteria. Moreover, this invention demonstrates that endophytes such as C. rosea can be combined with other beneficial bacteria, or fungi due to the fact that the 88-710 strain of C. rosea is not toxic to such organisms i.e. does not exude toxic metabolites, antibiotics or enzymes as to other bio-control fungi or mycoparasites. One of the key feature benefits of inoculants is that true inoculants can be ad-mixed together for mutual benefit, unlike bio-control agents.
This invention will show that some endophytes such as C. rosea strain 88-710, are more specialized in that they possess the ability to penetrate inside root tissues, leaves, stems, etc of plants and have direct access to organic compounds present in the apoplast”. Once inside the apoplast of plant tissues, endophytes such as C. rosea can survive and exude volatile exudates/compounds that promote plant health as well induce the plant to protect itself from various stress factors via IIR (inoculant induced resistance) e.g. as disease infections (botrytis, fusarium, Pythium, etc.) and or insect/mite attack. C. rosea has the unique inoculant feature/benefit of living within living and or senescent plant tissue and either remaining dormant or using that tissue as food to provide plants with nutrition even though the plant tissue may have been wounded by a disease, mite or insect. All of these are hitherto new, inventive, novel and useful features for endophytes acting as inoculants.
As indicated, Endophytes may also promote growth directly by the fixation of nitrogen alone or in combination with nitrogen fixing bacteria and or solubilization of minerals such as phosphorus, and provide the production of non-toxic siderophores that solubilize or sequester iron and other key micronutrients (manganese, zinc, etc.). The above inventive, novelty, application and new teachings of this invention show the endophyte inoculant benefits of promoting plant vigor, health and growth. In order for endophytes e.g. C. rosea, strain 88-710 to exhibit such benefits, the endophyte e.g. C. rosea needs to be present in sufficient quality/quantity and in a useful bio-available emulsifiable form as an effective and rapid ‘site occupier’ i.e. stable, robust spores in a deliverable formulation. The claims of this invention provide those rapid “site occupier” characteristics.
This invention also describes a novel, inventive and use application (improved sporulation and stability), for the production of an endophyte e.g. C. rosea that optimizes manufacture, concentration and thereby the performance of an endophyte product, for various uses as an inoculant(s).
Moreover naturally occurring endophytes such as C. rosea, strain 88-710 (which was originally identified from some 1400 different naturally occurring field isolates (samples) from Ontario strawberry fields, would have remained a relatively ineffective obscure endophyte/inoculant was rendered effective by this invention to provide the above inoculant plant health benefits and induced plant disease/insect/mite reduction feature/benefits by being available in a practical, useful and effective (as a site occupier) product formulations against major plant pathogens/insect pests e.g. Botrytis cinerea (grey mould). Without these key production and physical traits, endophytes such as C. rosea might remain relatively ineffective as inoculants.
This invention is also particularly applicable where wounding during pruning, cuttings, wounding, root trimming, transplanting/transplants and the like occurs.
The use of endophytes as inoculants, such as C. rosea, has the potential to offer a number of feature/benefits to help counter inadequacies, periodic failures and concerns associated with present pest control and costly agronomic cultural practices. These include cultural and sanitation measures, regulation of the microclimate, and reduce the heavy dependency on synthetic fungicides/insecticides with consequences such as fungicidal/insecticidal resistance, environmental and human exposure/loading and the general reduction of production costs, the reduction of energy needs/costs (hence reduction of greenhouse gases/contaminants), time to market, and enhancement of crop quality.
A patent has been granted e.g. U.S. Pat. No. 6,495,133 Dec. 17, 2002 using a certain strain coded ATCC # 74447 of Gliocladium roseum as a biocontrol agent for controlling diseases caused by fungal pathogens e.g. Fusarium, Ascochyta, Pythium, Rhizoctonia in plants for treatment of seeds, soil or plants (pea, bean, canola, wheat, barley, horticultural and ornamental plants). Patents have also been granted e.g. U.S. Pat. No. 6,475,566 Nov. 5, 2002 or U.S. Pat. No. 5,344,252 Jul. 9, 1996 for the protection of lumber against sapstains or fungi. However, none of these patents identified the novel usefulness of this invention for Clonostachys rosea, strain 88-710 or endophytes as beneficial inoculants for promoting plant vigor, health and growth in plants in the absence of disease or insect pressures or this combined with induced plant resistance to environmental stresses by plants themselves.
In particular, outlined in this invention, the need to identify and provide the correct physical properties to an endophyte for a high level of inoculant “rapid site occupation” to ensure performance in plants that in turn provides for improved leaf, stem and root growth and thereby reducing stress factors (environmental as well as cultural) of plants.
There have also been examples of other patents granted for endophytes e.g. U.S. Pat. No. 6,815,591 Nov. 9, 2004, or U.S. Pat. No. 5,723,720 Mar. 3, 1998, that provide plants via seed with imported insect resistance and drought tolerant traits into plants. However, again these patents do not document inventiveness, novelty, or usefulness regarding enhanced plant growth, overall plant health enhancement or these traits combined with plant inoculant resistance to stresses e.g. diseases or the ability to enhance endophyte product formulations to provide added value inoculant feature/benefits to allow plants themselves to resist attack from pests.
Other patents e.g. U.S. Pat. No. 4,550,527 Nov. 5, 1985 describe methods using special soil mixtures and containers of how to best infect roots of plants with beneficial mycorrhizal fungi for the purposes of improving plant health but these methods are not related to endophytes or to specific endophytes such as C. rosea esp. strain 88-710 or to specific feature/benefits of site inoculation and growth enhancement.
The present invention overcomes drawbacks in the prior art and teaches something new for information in the public domain. The drawbacks are overcome by a combination of the features of the main claims. The sub-claims disclose further advantageous embodiments of the invention and may also overcome drawbacks in the prior art. The present invention provides for many of the key feature/benefits the marketplace is seeking e.g.:                (1) Natural Endophyte products such as C. rosea, strain 88-710 that can act as plant inoculants that provide unique feature/benefits for the promotion of plant vigor, health, growth and yield including stimulation of legume plants to produce nodules for fixation of nitrogen (until this invention an unknown mode of action for fungal endophytes)        (2) Natural crop enhancement products that can increase yields, quality and reduce growing periods amidst environmental and cultural stresses e.g. drought, better utilization of nutrients, thus lowering energy costs and consequences of inefficient use of energy.        (3) Little or no risk of increased environmental or contaminate hazards due to the use of conventional agrochemicals/pesticides or fertilizers.        (4) Effective production of a natural bio agent in a stable useful formulation(s) that imparts/enhances unique physical ‘site inoculant occupation’ within plant tissues that can be organically certified for a growing market need i.e. OMRI certification, that in turn        (5) Imparts growth and plant protectant properties as a plant inoculant resistance effect against diseases, wounds or in a combined action for improved crop production that is unlikely to develop into “lack of performance resistance” as do chemicals pesticides.        (6) Provide natural wound healing benefits as an inoculant that can in turn in suitable formulations (that contain natural salts/emulsifiers) can provide plant protective benefits against a wide range of plant diseases, insects and mites e.g. powdery mildew, two-spotted mites, aphids.        
There is strong market interest to have as agronomic tools bio agents that are naturally occurring for use in improving crop production and quality of life e.g. quality, yield and time to market that can qualify for organic certification e.g. OMRI.
The economic impact for the use of this invention is documented in terms of feature/benefits. Solutions to the recurring problem of plant pathogens have been explored for decades. As particular crops become more abundant, and the area of land allocated for agriculture expands, or as greenhouse needs expand, there is an inherent need to employ more efficient and effective agronomic practices, preferably those beneficials provided by Mother Nature herself and that occur naturally in our global environment.
As a result of increasing demand for crop production, farmers must often compromise their cultural practices by planting crops on sub-optimal land, or by increasing the frequency at which crops are planted in a specific location. In doing so, crop nutrients are depleted, a microbial shift occurs and specific crop pathogens, especially soil-borne or seed-borne pathogens, become more prevalent. Accordingly, it is increasingly difficult to sustain the health and productivity of crops. It has been well documented that the fungal species C. rosea occurs most abundantly in virgin soils, wherever such soils can still be found around the world; in depleted or overworked soils C. rosea and other beneficial endophytes has been gradually eroded and are nearly or completely absent from soils thus reducing soil productivity and hampering farmers from producing productive crops e.g. in some African soils. The need for improved soil conditioning and the beneficial chain effect on global economies could be massive.
California alone has a $1.2 billion a year fruit industry; the strawberry crop alone is estimated to be worth over $400 million annually but is under pressure to minimize water/energy inputs and produce more “organic” crops to minimize pesticide usage.
There is increasing pressure in Europe to use only organically approved crop production tools amidst a lack of such tools.
In the instance of field peas as one example of a field “pulse” crop in Canada, there were approximately 1,000,000 hectares/year of field pea grown in western Canada in 1999-2005, estimated at a total farm value of over $600+ million and other oil seed crops such as Canola when acreages have increased to over 15 million acres planted annually amidst growing input problems.
In recent years, the field pea, lentil and other specialty crops of western Canada have been most affected by soil-borne or seed-borne diseases, as well as the Ascochyta complexes of root and foliage. Known as PRRC (pea root rot complex) diseases, the soil-borne and seed-borne diseases are most commonly caused by the pathogens Fusarium solani f.sp. pisi, Fusarium oxysporum f.sp. pisi, Mycosphaerella pinodes, Rhizoctonia solani, Sclerotina sclerotiorum, Aphanomyces euteiches, Alternaria alternata and Pythium spp. The incidence of PRRC diseases varies with year and location, while its severity is largely dependent on climate, crop rotation and cultural practices. The yield losses of pea crops alone, as a result of PRRC pathogens including foliar infection by Ascochyta complex, are frequently devastating with a conservative 15%-20% yield reduction translating into an approximate annual loss of $80+ million in areas of western Canada.
The rose and flower industry is estimated to be worth over $5 to $6 billion dollars (US) in North America at the grower level. The industry normally requires some 11-14 weeks turnaround time from cuttings to market shipment. The ability to produce a higher quality product with a reduction in some 10-20 days to market (as the technology in this invention provides) is a significant cost benefit to the flower/horticultural industry.
Greenhouse vegetable crops e.g. tomato, cucumber, peppers, etc in Canada alone are worth an estimated $1.0 billion and employ over 19,000 people. The US industry is valued at more than 10 times that. Losses to individual greenhouses e.g. a cucumber grower in a production area of 44,500 m2, crop productivity is frequently 10-15% lower in areas with diseased plants than where plants were generally healthy. This loss translates to more than $375,000 per year.